This invention relates to the extraction of bacterial antigens, particularly for immunoassays.
Bacterial antigens are bacterial substances which generate, or which immunospecifically react with, anti-bacterial antibodies. The presence of such an antigen in a sample is standardly detected by an immunospecific reaction with anti-bacterial antibodies.
Some bacterial antigens may be encrypted in larger cell structures which interfere with the immunospecific reaction or with its detection. Moreover, the sample may include other substances such as cell debris, mucus or other flora, which also can infere with the immunospecific reaction or with its detection.
Thus, it is important, e.g., preliminary to an immunoassay, to extract a bacterial antigen from cell structures and other sample components that surround it. This process is known as extracting the bacterial antigen.
Rosenstein, Published European Patent Application 153477, discloses a diagnostic test for group A streptococci in which an enzymatic extraction reagent releases strep A antigen from a swab, and agglutination with antibody immobilized on latex beads is detected.
A more customary rapid extractive medium is a combined solution of acetic acid and sodium nitrite. For example, Kholy et al. (1974) Applied Microbiology 28(5):836-839 and Hafez et al. (1981) J. Clin. Microbiol. 14(5):530-533 disclose a method of extracting protein antigens from streptococci in which NaNO.sub.2 and glacial acetic acid are added to a cell preparation. The extracted antigens are then subjected to immunoassay.
Unfortunately, the above-described nitrous acid extraction solution is highly unstable. It is apparent that the acetic acid must be kept separate from the nitrite until just before extraction takes place. Otherwise the instability of the resulting nitrous acid solution can defeat the extraction. Thus if the extractive materials are mixed prematurely there is a decay and the resultant solution becomes unsatisfactory after but a short delay interval.
In addition, the required separate packaging and mixing of the precursors is prone to leakage, inconvenient, time consuming, and liable to error.
Once the extraction is completed, the extractive medium may be neutralized, e.g., with NaOH as disclosed in Kholy et al. or Hafez et al. cited above, and the assay proceeds in conventional fashion.
Accordingly, it is an object of the invention to facilitate the extraction of test substances, as well as the assay of test materials. A related object is to facilitate immunoassays, particularly enzyme immunoassays.
Another object of the invention is to facilitate the extraction of nitrogenous substances, such as streptococcal cell walls. A related object is to completely eliminate the need for separate packaging of liquid acid or precursor prior to extraction.
Still another object of the invention is to reduce the amount of handling required for the extraction of antigens before performing enzymatic assays.
Yet another object of the invention is to eliminate the need for combining sodium nitrite with a liquid activator such as acetic acid. A related object is to eliminate any adverse consequences from the premature combination of an extractive agent with a liquid activator.